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Biomedical subjects

D Troyer

Publications and source records attributed to D Troyer.

At least 19 recordsLinked to original sources
PubMed2002

Diabetic nephropathy with interstitial nephritis presenting with a false-positive anti-GBM antibody.

A 56-year-old male with DM and HTN presented with flank pain and nausea. Review of systems was negative, physical examination was notable for mild hypovolemia and laboratory revealed BUN 51 mg/dl, creatinine (Cr) 5.1 mg/dl (baseline 1.5), Westergren ESR 122 mm/h, fractional excretion of sodium 0.2% and UA positive for blood and protein. Despite volume resuscitation the Cr continued to rise. Urine sediment analysis revealed granular casts, renal tubular epithelial cells and a negative Hansel's stain. Hemodialysis was initiated with Cr 13.7 mg/ dl for dyspnea and dysgeusia. Subsequent laboratory data revealed 2 separate positive anti-GBM antibody titers and prednisone therapy was initiated. Renal biopsy was performed for further diagnostic, therapeutic and prognostic information and demonstrated interstitial nephritis with linear IgG and albumin deposition consistent with diabetic nephropathy. Follow-up antibody titers were negative. prednisone was discontinued and Cr stabilized with conservative therapy. Anti-GBM antibody disease is characterized by circulating IgG antibodies directed against the glomerular basement membrane, specifically the alpha-3 (IV) collagen chain. Anti-GBM nephritis is a rapidly progressive, isolated glomerulonephritis in association with circulating anti-GBM antibodies. A positive immunofluorescence (IF) test is considered diagnostic in the appropriate clinical setting. Therapies include immunosuppressive agents to suppress new antibody production and plasmapheresis to eliminate circulating antibodies. Anti-GBM antibody is not rapidly cleared by steroid therapy and the recovery of renal function is rare if initiation Cr is greater than 7 mg/dl. This case demonstrates that the current ELISA for alpha-3 (IV) collagen is not pathognomonic for anti-GBM nephritis and that renal biopsy with IF for IgG and albumin may be indicated to prevent administration of potentially toxic treatment.

Anti-Glomerular Basement Membrane Disease↗
PubMed2001

Demonstration of the extrinsic coagulation pathway in teleostei: identification of zebrafish coagulation factor VII.

It is not known whether the mammalian mechanism of coagulation initiation is conserved in fish. Identification of factor VII is critical in providing evidence for such a mechanism. A cDNA was cloned from a zebrafish (teleost) library that predicted a protein with sequence similarity to human factor VII. Factor VII was shown to be present in zebrafish blood and liver by Western blot analysis and immunohistochemistry. Immunodepletion of factor VII from zebrafish plasma selectively inhibited thromboplastin-triggered thrombin generation. Heterologous expression of zebrafish factor VII demonstrated a secreted protein (50 kDa) that reconstituted thromboplastin-triggered thrombin generation in immunodepleted zebrafish plasma. These results suggest conservation of the extrinsic coagulation pathway between zebrafish and humans and add credence to the zebrafish as a model for mammalian hemostasis. The structure of zebrafish factor VIIa predicted by homology modeling was consistent with the overall three-dimensional structure of human factor VIIa. However, amino acid disparities were found in the epidermal growth factor-2/serine protease regions that are present in the human tissue factor-factor VIIa contact surface, suggesting a structural basis for the species specificity of this interaction. In addition, zebrafish factor VII demonstrates that the Gla-EGF-EGF-SP domain structure, which is common to coagulation factors VII, IX, X, and protein C, was present before the radiation of the teleosts from the tetrapods. Identification of zebrafish factor VII significantly narrows the evolutionary window for development of the vertebrate coagulation cascade and provides insight into the structural basis for species specificity in the tissue factor-factor VIIa interaction.

Amino Acid Sequence↗
PubMed2001

Xenotransplantation of bovine bone marrow stromal cells into pig fetuses: incorporation into skeletal muscle.

Bone marrow stromal cells are multipotent and have been shown to differentiate into a wide variety of mesenchymal cell types in vivo. In this study we tested the ability of bovine bone marrow stromal cells to contribute to the development of porcine skeletal muscle tissue. Fetal pigs were injected early in gestation with bone marrow stem cells originating from slaughtered steers. After approximately forty days of development the fetuses were harvested and sections of their skeletal muscle were analyzed for the presence of bovine cells. PCR was used to detect bovine DNA present in DNA extracted from the fetal pig skeletal muscle. We also used a PRINS (Oligonucleotide Primed In- Situ Synthesis) protocol to confirm the presence of bovine cells within the porcine skeletal muscle tissue sections. The results of both assays indicate that bovine bone marrow stromal cells can participate in the development of porcine skeletal muscle. This study helps to demonstrate the potential that bone marrow stromal cells have to contribute to advances in animal biotechnology and medicine.

Animals↗
PubMed2001

Life span is prolonged in food-restricted autoimmune-prone (NZB x NZW)F(1) mice fed a diet enriched with (n-3) fatty acids.

Moderate food and/or energy (calorie) restriction delays age-related immune dysfunction and prolongs life span in multiple animal models. The amount and type of dietary fatty acids can also profoundly affect life span. Marine-derived fish oils contain (n-3) fatty acids, which have potent anti-inflammatory properties. We therefore examined the influence of food restriction (40% overall reduction in intake of all dietary components) combined with substitution of fish oil for corn oil in a factorial design. Autoimmune-prone (NZB x NZW)F(1) (B/W) mice, which develop fatal autoimmune renal disease, were used. The food-restricted/fish oil diet maximally extended median life span to 645 d (vs. 494 d for the food-restricted corn oil diet). Similarly, fish oil prolonged life span in the ad libitum-fed mice to 345 d (vs. 242 for the ad libitum/corn oil diet). Increased life span was partially associated with decreased body weight, blunting renal proinflammatory cytokine (interferon-gamma, interleukins-10 and -12 and tumor necrosis factor-alpha) levels and lower nuclear factor-kappaB (NF-kappaB). Reductions in NF-kappaB were preceded by enhanced superoxide dismutase, catalase and glutathione peroxidase activities. These findings demonstrate the profound additive effects of food restriction and (n-3) fatty acids in prolonging life span in B/W mice. These observations may have additional implications in the management of obesity, diabetes, cancer and/or the aging process.

Analysis of Variance↗
PubMed2000

Differences in the effects of HMG-CoA reductase inhibitors on proliferation and viability of smooth muscle cells in culture.

We investigated the influence of lovastatin, simvastatin and pravastatin on proliferation and viability of vascular smooth muscle cells (SMC) in vitro and studied the effects of lovastatin on a mouse SMC line transgenic for a temperature-sensitive mutant of SV40 large T antigen (TAg), known to inhibit the function of p53 and pRb family members. We found that lovastatin and simvastatin inhibited cell proliferation by provoking G0/G1 phase arrest with concomitant depression of the proliferation antigen Ki-67/MIB-1. Lovastatin at high concentrations of 20 micromol/l caused cell death in the presence of serum but not under serum starved conditions, which was verified on the basis of increased DNA strand breaks, decreased DNA content and morphological alterations seen by electron microscopy. Cell death was also found for simvastatin, whereas pravastatin did not exhibit antiproliferative or cytotoxic effects. Mouse SMC transgenic for TAg did not show any impaired sensitivity to the antiproliferative and cell death inducing effect of lovastatin, but both effects could be antagonized by the supplementation of mevalonate. The data indicate that antiproliferative and cytotoxic effects of lovastatin are caused by the using up of products of mevalonate metabolism and do not require the presence of p53 or pRb.

Animals↗
PubMed2000

Risk perceptions among patients and their relatives regarding prostate cancer and its heredity.

We performed a qualitative study to examine how prostate cancer (PC) patients and their spouses and relatives take into account family history when considering susceptibility to PC. Semi-structured interviews were conducted with 20 participants. All interviews were tape-recorded, transcribed and content analyzed. Patients' and spouses' views concerning the seriousness of PC were different. Wives viewed PC as a serious disease because it has affected their marital relationships; patients found PC to be less serious because it can be treated. All participants viewed PC as a male disease that can be passed on from fathers to sons. Furthermore, participants were aware of PC clustering in their families. However, this awareness did not encourage (healthy) male relatives to seek early detection. Additionally, participants perceived age, high-fat diet, and less exercise as important risk factors, while socioeconomic status, ethnic origin and a family history of PC were viewed as less important. We recommend that health educators pay special attention to these findings when planning to teach patients, spouses and their relatives about PC, its heredity and risks. Prostate Cancer and Prostatic Diseases (2000) 3, 176-185

Journal Article↗
PubMed2000

Isolation of thirty-one new porcine microsatellites from a microsatellite enriched microdissected chromosome 8 library.

Chromosome 8 (SSC8) is an important one in the swine genome because it has been shown to harbor several economically important quantitative trait loci (QTL). The entire porcine chromosome 8 was microdissected and amplified by degenerate oligonucleotide primer (DOP) PCR. The PCR product was then enriched for (CA)n microsatellites by hybridization to a biotinylated CA repeat oligonucleotide and captured by streptavidin-coated magnetic beads. The captured DNA was cloned into a TA cloning vector. Screening with an isotopically labeled CA oligonucleotide probe resulted in the isolation of 31 informative and polymorphic microsatellite clones. Seventeen of those were mapped to chromosome 8, 12 to chromosome 15, 1 to chromosome 10 and 1 to chromosome X. The markers were all placed on the USDA-MARC porcine genetic linkage map.

Animals↗
PubMed1999

Cloning and characterization of the gene for a new epithelial beta-defensin. Genomic structure, chromosomal localization, and evidence for its constitutive expression.

Mammalian beta-defensins are endogenous cysteine-rich peptide antibiotics that are produced either by epithelial cells lining the respiratory, digestive, and urogenital tracts or by granulocytes and macrophages. A growing body of evidence has implicated these peptides in host defense, particularly mucosal innate immunity. We previously reported the cloning of the full-length cDNA for a porcine beta-defensin (pBD-1), which was found to be expressed throughout the airway and oral mucosa. Here, we provide the structural organization of the pBD-1 gene, showing that the entire gene spans approximately 1.9 kilobases with two short exons separated by a 1.5-kilobase intron. Fluorescence in situ hybridization mapped the pBD-1 gene to porcine chromosome 15q14-q15. 1 within a region of conserved synteny to the chromosomal locations of human and mouse alpha- and beta-defensins. We also provide several independent lines of evidence showing that the pBD-1 gene is expressed constitutively during inflammation and infection, despite its resemblance to many inducible epithelial beta-defensins in amino acid sequence, genomic structure, and sites of expression. First, stimulation of primary porcine tongue epithelial cells with lipopolysaccharide, tumor necrosis factor-alpha, and interleukin (IL)-1beta failed to up-regulate the expression of pBD-1 mRNA. Second, pBD-1 gene expression was not enhanced in either digestive or respiratory mucosa of pigs following a 2-day infection with Salmonella typhimurium or Actinobacillus pleuropneumoniae. Last, direct transfection of the pBD-1 gene promoter into NIH/3T3 cells showed no difference in reporter gene activity in response to stimulation by lipopolysaccharide and IL-1beta. The constitutive expression of pBD-1 in airway and oral mucosa, which is consistent with a lack of consensus binding sites for nuclear factor-kappaB or NF-IL-6 in its promoter region, suggests that it may play a surveillance role in maintaining the steady state of microflora on mucosal surfaces.

3T3 Cells↗
PubMed1999

Identification and characterization of zebrafish thrombocytes.

To analyse primary haemostasis in the zebrafish we have identified and characterized the zebrafish thrombocyte by morphologic, immunologic and functional approaches. Novel methods were developed for harvesting zebrafish blood with preservation of thrombocytes, and assaying whole blood adhesion/aggregation responses in microtitre plates. Light and electron microscopy of the thrombocyte illustrated morphological characteristics including the formation of aggregates, pseudopodia, and surface-connected vesicles analagous to the platelet canalicular system. Immunostaining with polyclonal antisera versus human platelet glycoproteins demonstrated the presence of glycoprotein Ib and IIb/IIIa-like complexes on the thrombocyte surface. Whole blood assays for adhesion/aggregation and ATP release showed ristocetin-induced adhesion without ATP release, and platelet agonist (collagen, arachidonic acid) induced aggregation with ATP release. Blood harvested from zebrafish treated with aspirin demonstrated inhibition of arachidonic acid induced aggregation and agonist induced ATP release, consistent with at least partial dependence on an intact cyclo oxygenase pathway. The combined morphologic immunologic and functional evidence suggest that the zebrafish thrombocyte is the haemostatic homologue of the mammalian platelet. Conservation of major haemostatic pathways involved in platelet function and coagulation suggests that the zebrafish is a relevant model for mammalian haemostasis and thrombosis.

Animals↗
PubMed1997

Effects on DNA integrity and apoptosis induction by a novel antitumor sesquiterpene drug, 6-hydroxymethylacylfulvene (HMAF, MGI 114).

6-Hydroxymethylacylfulvene (HMAF, MGI 114) is a new alkylating antitumor sesquiterpenoid with promising and often curative antitumor activity in vivo. This study examined the ability of the drug to damage cellular DNA, induce apoptosis, and affect the cell cycle of CEM human leukemia cells. No bifunctional lesions, interstrand DNA cross-links or DNA-protein cross-links were seen (by alkaline sedimentation and K+/SDS precipitation, respectively) when using up to 50 microM HMAF. The drug possibly formed some monoadducts, as DNA from drug-treated cells impeded primer extension by Taq polymerase, although only partial inhibition was seen even at 200 microM HMAF. HMAF also induced secondary lesions in cellular DNA, single-strand breaks that were detectable (by nucleoid sedimentation and alkaline sucrose gradient analysis) after a 4-hr treatment at HMAF levels as low as 2 microM, comparable to the growth inhibition IC50 value (1.7 microM). A post-treatment incubation of cells in drug-free medium generated substantial amounts of DNA double-stranded fragments of several kbp, suggesting apoptotic fragmentation (>30% of total DNA following treatment with 20 microM HMAF and a 17-hr post-treatment incubation). Chromatin condensation (by ultrastructural analysis) and induction of sub-G1 particles and apoptotic strand breakage (by multiparametric flow cytometry) confirmed induction of apoptosis by HMAF. HMAF preferentially inhibited DNA synthesis (IC50 approximately 2 microM), which is consistent with an S phase block, observed by cell cycle analysis. The pattern of apoptotic DNA fragmentation, inhibition of DNA synthesis, and blockage in the S phase suggests that these events play a role in the antiproliferative activity of HMAF.

Antineoplastic Agents↗